The present invention relates to plant cell transformation in which genetic material is inserted into plant cells to modify resulting plants, and in particular, the invention relates to an apparatus for collecting embryonic tissue from seeds that may be used for such transformation.
The genetic transformation of plants may be used to develop crops with improved yield, insect and disease resistance, herbicide tolerance, and increased nutritional value. In such transformation, new genes are introduced into the chromosomal material of existing plant cells. Various methods have been developed for transferring genes into plant tissue including high velocity microprojection, microinjection, electroporation, direct DNA uptake and, Agrobacterium-mediated gene transformation.
Once the gene is successfully introduced into the chromosomal material of the plant cells, new inheritable germ line tissue must be developed (e.g., seeds) so that the new plant may be propagated. One way this may be done is by selecting only cells that have accepted the new gene and culturing the callus of these cells into a new viable plant. The time required to develop a plant from a single cell is lengthy.
Shortened development times may be obtained by directly treating meristematic tissue of a preformed plant embryo. The meristematic tissue is formative plant tissue of cells that will differentiate to produce different plant structures including the seeds or germ line tissue. A number of plant embryos may be treated and selection or screening techniques used later to determine which of those plants have incorporated the new genetic information into their germ line tissue.
U.S. Pat. No. 6,384,301 assigned to the assignee of the present invention and hereby incorporated by reference describes a method of genetically transforming soybeans (Glycine max) using Agrobacterium mediated gene transfer directly on the meristematic cells of soybean embryos. In this procedure, the seeds are soaked to initiate germination. After germination has begun, the embryo is excised from the seed and the primary leaf tissue removed to expose the meristem of the soybean embryo. The meristem is formative plant tissue that will differentiate to give rise to different parts of the plant.
Although seeds are inexpensive, the considerable labor involved in excising the embryos, transferring the genetic material into the embryos, and cultivating the embryos makes it desirable to reduce damage to the embryo that could result in this effort being applied to tissue that is ultimately non-viable. For this reason, the excision of plant embryos is performed by hand.
In the manual process, surface sterilized seeds are aseptically handled one at a time with gloved hands. They are oriented in a manner as to eject the seed coat with applied force. Then the cotyledons are separated and removed leaving the seed embryo. The embryonic leaves are removed near the area of the primary meristem. Recovery of viable embryos for genetic transfer is less than 100% even with this hand method and may be as little as 70% with high quality seeds.
Bacterial contamination of the embryos after excision is a significant concern. Manual excision of the embryos allows early separation of the seed coat from the remainder of the seed to prevent contamination of the embryo with bacteria found on the seed coat, which normally protects the embryo.
Skilled personnel performing manual excision can often recognize abnormal embryos at the time of excision and discard them, substantially improving downstream yields.
Despite the advantages of manual excision, individual separation of each plant embryo from its seed is extremely labor intensive and stands as a barrier to a scaling up of the transformation process in which, typically, many plants must be treated to yield a successful few transformations.
What is needed is a process that can significantly increase the availability of transformable embryos without unacceptably increasing total costs of transformation, the latter which will rise if damage to embryos or bacterial contamination of the embryos causes fruitless cultivation of large numbers of non-viable embryos.